The present invention relates to a method of measuring the pH of a cell culture solution, and also to an accurate pH measuring method and apparatus in which an influence of a predetermined component such as a cell proliferation factor of fetal bovine serum (FBS) that is contained in a medium and that has an unknown concentration is corrected.
In order to grow and proliferate cells, the pH of a culture solution containing the cells must be within a range suitable for proliferation. During preparation or storage of such a cell culture solution, however, carbon dioxide which is contained in the cell culture solution is released, and the pH is increased, so that the pH is often deviated from the proliferation suitable range.
Therefore, the pH is measured by a method in which, for example, the color change of phenol red that is contained in a cell culture solution is visually checked, or that in which the measurement is performed while pH electrodes are immersed in a cell culture solution. However, these methods have the following problems.
In the case where the color change of phenol red contained in a cell culture solution is visually checked, an erroneous check may be caused. This is because an error often occurs in a visual check, and serum contained in a cell culture solution, such as fetal bovine serum exhibits a yellow color, and hence a check result is sometimes confused by the coloration.
By contrast, in the case where pH electrodes are immersed in a cell culture solution, when the pH electrodes are not sufficiently sterilized, contamination due to bacteria or the like may occur.
As a method of measuring the pH of a cell culture solution which is free from problems such as an error due to a visual check, and contamination in the case where pH electrodes are used, the following method is known (see JP-B-06-34754).
FIG. 1 is a block diagram showing a pH measuring apparatus which can measure the pH of a medium solution.
In the pH measuring apparatus of the example, a light emitting element 1 is driven by a driving circuit 3 so as to emit light beams by using an electric power supplied from a battery 4.
Although the light emitting element 1 is shown as one element in FIG. 1, the light emitting element is actually configured by a plurality of elements respectively emitting light beams of different wavelengths. More specifically, the light emitting element 1 is configured by at least four elements such as: an element emitting a light beam of 411 nm band which is a peak of the absorbance corresponding to fetal bovine serum (FBS); that emitting a light beam of 430 or 560 nm band indicating a peak of the absorbance of phenol red (pH indicator); that emitting a light beam of 367 or 479 nm band indicating a value to which the absorbance is unchanged and converged irrespective of a change of the pH; and that emitting a light beam of 700 nm band in which, for example, fetal bovine serum and phenol red do not exhibit absorbance. For example, LEDs are preferably used as these elements.
The light beams emitted from the light emitting element 1 are transmitted through a solution 5 in which the pH is to be measured, and then received by a light receiving element 6, to be converted to electric signals. Although the light receiving element 6 is shown as one element in FIG. 1, the light receiving element is actually configured by a plurality of elements respectively corresponding to the elements constituting the light emitting element 1. Photodiodes are preferably used as these elements. In the case where the light emitting element 1 is configured by four LEDs, namely, the light receiving element 6 may be configured by four photodiodes. In place of the mode where the light receiving element 6 receives light beams which have been transmitted through the measurement solution 5, the light receiving element may be disposed so as to receive light beams reflected from the measurement solution 5.
The signals which are converted in the light receiving element 6 are amplified by an amplifier 7, and then distributed by a multiplexer 8 to filters 9 which correspond to the light wavelengths, respectively.
The signals distributed to the filters 9 are filtered by the filters 9 to reduce noise components, digitized by an A/D converter which is not shown, and then supplied to a processing section 10. For example, the processing section 10 is configured by a calculation processing device such as a CPU, a storage device, etc.
JP-B-06-34754 describes a related-art method of measuring the pH of a cell culture solution as follows.
The method of measuring the pH of a cell culture solution is a method in which the pH is measured based on absorption of visible light in a cell culture solution configured by: a cell culture medium; serum; and an indicator having two or more kinds of absorption peaks in the wavelength region of visible light, wherein, based on a linear relationship between the pH and the logarithms of absorbances at two wavelengths of absorption peaks that are obtained by transmitting visible light through a cell culture solution in which the pH is known, the value of the pH is obtained from the value of the logarithm of a ratio of absorbances of absorption peaks that are measured in a cell culture solution specimen in which the pH is not known.
In the cell culture solution, usually, phenol red for detecting a change of the pH is contained at a low concentration which does not harm cells. In the visible light range, at such a low concentration, phenol red has peaks in the vicinities of 430 to 440 nm and 560 nm, and an isosbestic point at 480 nm. In a pH range of 6.8 to 7.6 where cells can grow, as the pH is further lowered, the absorption peak in the vicinity of 430 to 444 nm is more increased, and that in the vicinity of 560 nm is more decreased. When absorption due to only the phenol red is obtained, by taking a ratio of absorption in the vicinity of 430 to 440 nm to that in the vicinity of 560 nm, the plot shows one curve, and the pH of the culture solution can be calculated from a ratio of the two peaks.
In the related-art optical pH measurement, moreover, the zero level measurement is performed with a sample blank prior to measuring the sample, and the absorption levels of the blank are then subtracted from the sample reading to provide the net absorbance of the sample. The vicinity of the wavelength (700 nm) which shows very little absorption by the indicator is chosen as the third wavelength, and is used as means for tracking changes in the zero level. Changes in the absorption level of this wavelength channel are indicative of changes in the zero level. Therefore, the other two wavelengths which are used in the measurement are zero corrected based on the changes measured at this third wavelength.
In the related-art method of measuring the pH of a cell culture solution disclosed in JP-B-06-34754, only phenol red is contained in the cell culture solution. In a cell culture solution, however, not only phenol red, but also, for example, fetal bovine serum (FBS) is sometimes contained as a proliferation factor for proliferating cells.
The optical absorbance characteristic of fetal bovine serum (FBS) is largely different from that of phenol red, and hence a measurement value obtained in the pH measuring method disclosed in JP-B-06-34754 cannot be used as it is. In the pH measuring method disclosed in JP-B-06-34754, particularly, a linear relationship between the logarithms of absorbances and the pH cannot be determined unless the concentration of the serum contained in the medium is known before the measurement. In a medium containing, for example, serum the concentration of which is unknown, therefore, the pH cannot be correctly measured.